Direct voltammetry of cytochrome c at trace concentrations with nanoelectrode ensembles

PTCA(PART B: CHEM. ANAL.)工作商报DOI ： 10.11973/lhjy-hx202201006基于纳米Ti02和TiN为工作电极的 循环伏安法测定过氧化氢的含量

黄章烤，王琳玲，赵峰鸣—(浙江工业大学化学工程学院，杭州310032)

摘要：制备了纳米Ti〇2、TiN材料，将其作为工作电极，建立了循环伏安法测定过氧化氢含量 的方法。将钦片置于含10.0 g氟化铵、0.6 g脲、24 mL 30%(质量分数）过氧化氢溶液、24 mL硝酸 组成的抛光液中进行下表面抛光处理，再加入15 mL丙酮、15 mL无水乙醇、15 mL水，超声处理 15 min后，得到光亮钦片。以光亮钛片为阳极，普通钛片为阴极，在不同电解质[Ti02粗糙膜对应 的电解质为1.0 mol . L_1硫酸溶液；Ti02纳米管为50 mL丙三醇、50 mL水、0.2 mol • L—1硫酸 溶液、0.5%(质量分数）氟化钠溶液；Ti02纳米孔为100 mL乙二醇、1 mL水、0.38%(质量分数）氟 化铵溶液]中，采用阳极氧化法，得到不同形貌大小的Ti02粗糙膜、Ti02纳米管、Ti02纳米孔；再 通过氨气热还原，得到TiN粗糙膜、TiN纳米管、TiN纳米孔。以Ti02和TiN材料为工作电极， 铂片为辅助电极，Ag/AgCl为参比电极，将三电极体系置于磷酸盐缓冲溶液（pH 7.0)中，采用循环 伏安法法测定过氧化氢含量。结果显示：TiN粗糙膜、TiN纳米管、TiN纳米孔电极的速率常数分 别为 2.39X10—6,3.03 X 10_6,6.40Xl(r6cm . s—1;以 Ti02 粗糙膜、TiN 粗糙膜、Ti02 纳米管、 TiN纳米管、Ti02纳米孔、TiN纳米孔为工作电极，过氧化氢的浓度在一定范围内与其对应的还原

峰电流呈线性关系，检出限（3"々）分别为23.30,14.29,19.9,10.6,16.9,5.02 /xmol • L一1;在磷酸盐 缓冲溶液（pH 7.0)中滴加50 pmol . L—1 H202溶液，采用计时电流法在一0.4 V外加电压下进行 测定，计算得TiN粗糙膜、TiN纳米管、TiN纳米孔响应电流的相对标准偏差（RSD，《 = 5)分别为 4.7%, 3.2%和 7.3%。关键词：TiN;过氧化氢；电化学性能；循环伏安法；计时电流法

随着社会科技的发展，绿色能源成为人类可持续发展的重要条件，而风能、太阳能等非可持性能源的开发和利用面临着间歇性和不稳定性的问题，这就催生了大量的储能装置，其中比较引人注目的包括太阳能电池、锂子电池和超级电容器等。

超级电容器作为一种新型化学储能装置，具有高功率密度、快速充放电、较长循环寿命、较宽工作温度等优秀的性质，目前在储能市场上占有很重要的地位，同时它也广泛应用于军事国防、交通运输等领域。

目前，随着环境保护观念的日益增强，可持续性能源和新型能源的需求不断增加，低排放和零排放的交通工具的应用成为一种大势，电动汽车己成为各国研究的一个焦点。

超级电容器可以取代电动汽车中所使用的电池，超级电容器在混合能源技术汽车领域中所起的作用是十分重要的，据英国《新科学家》杂志报道，由纳米花和纳米草组成的纳米级牧场可以将越来越多的能量贮存在超级电容器中。

随着能源价格的不断上涨，以及欧洲汽车制造商承诺在1995年到2008年之间将汽车CO2的排放量减少25%，这些都促进了混合能源技术的发展，宝马、奔驰和通用汽车公司已经结成了一个全球联盟，共同研发混合能源技术。

2002年1月，我国首台电动汽车样车试制成功，这标志着我国在电动汽车领域处于领先地位。

而今各种能源对环境产生的负面影响很大，因此对绿色电动车辆的推广提出了迫切的要求，一项被称为Loading-leveling(负载平衡)的新技术应运而生，即采用超大容量电容器与传统电源构成的混合系统“Battery-capacitor hybrid”(Capacitor-battery bank) [1]。

目前对超级电容器的研究多集中于开发性能优异的电极材料，通过掺杂与改性，二氧化锰复合导电聚合物以提高二氧化锰的容量[1、2、3]。

生瑜(是这个人吗？)等[4]通过原位聚合法制备了聚苯胺/纳米二氧化锰复合材料，对产物特性进行细致分析。

因导电高分子具有可逆氧化还原性能，通过导电高分子改性，这对于提高二氧化锰的性能和利用率是很有意义的。

第44卷第6期2021年6月核技术NUCLEAR TECHNIQUESV ol.44，No.6June2021水系锌离子电池正极材料碲化铋层间质子可逆输运的原位观测彭磊1,2,3王娟1,2,3何燕1,2,3杨科1,2,31（中国科学院上海应用物理研究所上海201800）2（中国科学院大学北京100049）3（中国科学院上海高等研究院上海同步辐射光源上海201204）摘要可充电水系锌离子电池因具备低成本、高安全性、无毒环保等优点备受关注，具有高比容量和工作电压的新型正极材料则是水系锌离子电池研究的热点之一，而碲化铋正是这样一种新兴材料。

本工作采用一步水热法剥离碲化铋（Bi2Te3）粉末获得稳定的碲化铋纳米结构材料，并首次探索将其作为正极材料应用于水系锌离子电池中。

扫描电镜和原子力显微镜测试结果均表明合成的碲化铋具有纳米片形貌，厚度仅为3~5nm。

为了进一步深入研究其反应机理，利用同步辐射原位X射线衍射技术，实时表征碲化铋纳米片正极在电池充放电过程中的微观结构变化，并实时观察到碲化铋纳米片反应过程中高度可逆的质子插层现象，证实了质子在碲化铋正极材料中的可逆输运特性。

关键词水系锌离子电池，碲化铋纳米片，原位同步辐射X射线衍射，可逆质子插层中图分类号TL99DOI:10.11889/j.0253-3219.2021.hjs.44.060103In-situ observation of reversible proton transport through Bi2Te3anodeof aqueous zinc-ion batteryPENG Lei1,2,3WANG Juan1,2,3HE Yan1,2,3YANG Ke1,2,31(Shanghai Institute of Applied Physics,Chinese Academy of Sciences,Shanghai201800,China)2(University of Chinese Academy of Sciences,Beijing100049,China)3(Shanghai Advanced Research Institute,Shanghai Synchrotron Radiation Facility,Chinese Academy of Sciences,Shanghai201204,China)Abstract[background]The rechargeable aqueous zinc-ion batteries have attracted increasing attention due to their low cost,high safety,non-toxicity and environmental protection.The exploitation of new anode materials with high specific capacity and working voltage is one of research hotspots of aqueous zinc-ion battery whilst bismuthtelluride is such an emerging materials.[Purpose]This study aims to observe the reversible proton transport in Bi2Te3anode of aqueous zinc-ion battery.[Method]First of all,bismuth telluride nanostructure was obtained by one-step hydrothermal exfoliating of bismuth telluride powder,and the bismuth telluride nanostructure was applied first time as anode material to aqueous zinc-ion battery.Then,both the scanning electron microscopy(SEM)and atomic force microscopy(AFM)were employed to oberve the nanosheets morphology and measure the thickness of synthesized中国科学挑战专项(No.TZ2018001)资助第一作者：彭磊，男，1995年出生，2018年毕业于南华大学，现为硕士研究生，研究领域为水系锌离子电池正极材料研究通信作者：王娟，E-mail：收稿日期：2021-02-25，修回日期：2021-03-23Supported by Science Challenge Project of China(No.TZ2018001)First author:PENG Lei,male,born in1995,graduated from University of South China in2018,master student,focusing on cathode materials of aqueous zinc-ion batteryCorresponding author:WANG Juan,E-mail:Received date:2021-02-25,revised date:2021-03-23核技术2021,44:060103Bi 2Te 3nanosheets.Finally,in-situ synchrotron radiation X-ray diffraction (XRD)technique was used to track the changes of structure of Bi 2Te 3nanosheets during the charge-discharge process of the battery.[Result]The thickness of synthesized Bi 2Te 3nanosheets measured by AFM is about 3~5nm,and a highly reversible proton intercalation disclosed by in-situ synchrotron radiation based XRD during the reaction process is responsible for the practical battery operation.[Conclusions]This study confirms the reversible transport properties of protons in bismuth telluride cathode materials.Key wordsAqueous zinc-ion battery,Bismuth telluride nanosheets,In-situ synchrotron radiation X-ray diffraction,Reversible proton intercalation可再生能源的开发是当今社会一直备受关注的热点问题之一。

Hans Journal of Chemical Engineering and Technology 化学工程与技术, 2020, 10(2), 111-118Published Online March 2020 in Hans. /journal/hjcethttps:///10.12677/hjcet.2020.102016Preparation of ZIF-67 DerivativeMicro-Nano Flower-Like Co3O4 Catalystand Its OER Catalytic PerformanceShunzheng Ren, Lijuan Feng, Shuo Yao*College of Chemistry and Chemical Engineering, Ocean University of China, Qingdao ShandongReceived: Mar. 2nd, 2020; accepted: Mar. 16th, 2020; published: Mar. 23rd, 2020AbstractUsing ZIF-67 as a precursor, micro-nano flower-like ZIF-67(f) was obtained based on the morpho-logical evolution of ZIF-67 based on ion-assisted solvothermal conditions, and micro-nano flow-ers-like Co3O4(f) was prepared in an air atmosphere by heat treatment. Electron microscope (SEM), transmission electron microscope (TEM), X-ray diffractometer (XRD), Fourier infrared spectro-meter (FT-IR), and gas adsorption instrument (BET) were used to characterize the morphology and structure of the material. The electrochemical performance of the material was tested using an electrochemical workstation, and the oxygen evolution reaction (OER) performance of the cat-alyst prepared at different temperatures was discussed. The results show that the electrocatalytic performance of the prepared flower-like Co3O4(f) is greatly improved compared with commercial Co3O4 and Co3O4(r). The micro-nano flower-like Co3O4(f) material prepared by calcination at 450˚C has the most excellent electrocatalytic performance. Its overpotential at a current density of 10 mA∙cm−2 is 390 mV, and the Tafel slope is 60 mV∙dec−1.KeywordsElectrocatalysts, MOFs, Co3O4, Oxygen Evolution Reaction, ZIF-67ZIF-67衍生物微纳米花状Co3O4催化剂的制备及其OER催化性能研究任顺政，冯丽娟，姚硕*中国海洋大学化学化工学院，山东青岛*通讯作者。

2010 年 2 月 Journal of Chemical Engineering of Chinese Universities Feb. 2010文章编号：1003-9015(2010)01-0071-05氨氮在两种电解质体系下的电化学氧化褚衍洋, 杨波, 李玲玲, 牟莹莹, 王绚, 王沙沙(青岛科技大学环境与安全工程学院, 山东青岛 266042)摘要：以钛基氧化物涂层材料(IrO2，RuO2，TiO2 / Ti)为阳极，石墨为阴极，考察了氨氮的直接电化学氧化与其在两种液相电解质体系下(存在氯离子与否)的氧化效果。

检测了氨氮氧化过程中亚硝态氮和硝态氮的生成量。

结果表明，当不存在氯离子时NH4+-N的电化学氧化可被忽略，但游离态氨氮(NH3-N)在约1.25V阳极电位下可发生直接氧化。

当电解质中存在氯离子时，有效氯的生成使氨氮的氧化效率显著提高，在碱性体系下氨氮的去除作用包括间接电化学氧化和直接电化学氧化，但以前者为主。

在两种电解质体系下均有少量硝态氮生成，而亚硝态氮仅在无氯离子体系下有少量生成。

硝态氮和亚硝态氮的存在说明部分氨氮可能发生了非有效氯的间接氧化。

本研究表明氨氮的电化学氧化机理与介质条件、阳极电位密切相关。

关键词:氨氮；钛基涂层阳极；电化学氧化；氯离子中图分类号：X703.1 文献标识码：AElectrochemical Oxidation of Ammonia Nitrogen in Two Aqueous ElectrolytesCHU Yan-yang, YANG Bo, LI Ling-ling, MU Ying-ying, WANG Xuan, WANG Sha-sha (School of Environment and Safety Engineering, Qingdao University of Science and Technology, Qingdao266042, China)Abstract: The direct electrochemical oxidation of ammonia nitrogen and its electrochemical oxidation effect in two aqueous electrolytes with/without chloride ion were investigated by using IrO2, RuO2 and TiO2/Ti as anode and graphite as cathode. The content of nitrite nitrogen and nitrate nitrogen produced in the electrochemical oxidation of ammonia nitrogen were determined. The results show that the electrochemical oxidation of NH4+-N in the electrolyte without chloride ion could be neglected, but the direct electrochemical oxidation of free NH3-N could take place when anodic potential is about 1.25 V. The present of chloride ion in electrolyte leads to the formation of effective chlorine, which could greatly enhance the oxidation efficiency of ammonia nitrogen, and in alkaline electrolyte, both indirect and direct electrochemical oxidation may account for the removal of ammonia nitrogen, but predominant mechanism is the former. The low content of nitrate nitrogen in above two aqueous electrolytes as well as the nitrite nitrogen in the electrolyte without chloride ion only were found, which indicate that the indirect oxidation of ammonia nitrogen caused by other species besides effective chlorine may be occurred. The study shows that the oxidation mechanism of ammonia nitrogen has close relation with electrolytic medium and anodic potential.Key words: ammonia nitrogen; titanium anode with coatings; electrochemical oxidation; chloride ion1引言电化学氧化作为一种高级氧化技术在污水处理领域的应用研究日益受到国内外关注。

第53卷第3期2021年3月Vol.53No.3Mar.,2021无机盐工业INORGANIC CHEMICALS INDUSTRY催化材料Doi：10.11962/1006-4990.2020-0232开放科学(资源服务)标志识码(OSID)微反应器水热法耦合制备纳米片状氧化铝王梦迪“,罗瑾周靖辉",于海斌吴巍",李晓云"(1.中海油天津化工研究设计院有限公司，天津300131；2.天津市炼化催化技术工程中心)摘要:以偏铝酸钠和硫酸铝为原料，通过一种高通量撞击流微反应器，提岀了将微反应法与老化、水热法高效集成制备纳米片状氧化铝的新工艺。

利用X射线衍射(XRD)、扫描电镜(SEM)、透射电镜(TEM)、BET法比表面积测定(BET)、热重-差热分析(TG-DTG)等方式对不同工艺耦合制备的产物进行了测试分析，研究了不同耦合方式对产物晶型、形貌、介孔结构等物理性质的影响。

结果表明:通过微反应器-水热法耦合技术能够制备粒径为30~100nm、厚度为2~5nm、纯度为99.7%以上的纳米片层状勃姆石(酌-AlOOH),经550益焙烧4h可制得同样形貌的酌-氧化铝(酌-Al J O i)。

通过不同工艺耦合能够调控氧化铝的形貌、介孔结构，为工业化制备片层状纳米氧化铝提供了很好的科研支撑。

关键词:高通量;微反应;水热;纳米片状氧化铝中图分类号:TQ131.1文献标识码:A文章编号：1006-4990(2021)03-0087-06Microreactor-hydrothermal coupling preparation of nano-flaky aluminaWang Mengdi1,2,Luo Jin1袁Zhou Jinghui1,2,Yu Haibin1,Wu Wei1,2,Li Xiaoyun1,2(1.CenerTech Tianjin Chemical Research and Design Institute Co.,Ltd.,Tianjin300131,China；2.Tianjin Refining Catalytic Technology Engineering Center)Abstract: A novel preparation process of nano-flaky alumina is proposed by using a high-throughput impinging stream microreactor with sodium metaaluminate and aluminum sulfate as raw materials,which efficiently integrates the mi­cro-reaction method with aging and hydrothermal method.The effect of different coupling methods on the physical properties of the product such as crystal shape,morphology and pore structure was studied by XRD,SEM,TEM,BET,TG-DTG and oth­er test methods.The flaky酌-AlOOH with purity of more than99.7%,particle size of30~100nm and thickness of2~5nm can be produced by microreaction-hydrothermal coupling technology.The酌-Al2O3with the same morphology can be obtained after calcination at550益for4h.The morphology and mesoporous structure of alumina powder can be regulated by the coupling of different processes.It provides theoretical support for the industrial production of nano-flaky alumina.Key words:high-throughput；microreaction；hydrothermal；nano-flaky alumina纳米氧化铝因具有优异的机械和化学性能,被广泛用于催化剂、复合增强材料、陶瓷材料、生物医学材料、半导体材料等。

UNIT7.32 Uncompensated Polychromatic Analysisof Mitochondrial Membrane PotentialUsing JC-1and Multilaser ExcitationSara De Biasi,1Lara Gibellini,1and Andrea Cossarizza11Department of Surgery,Medicine,Dentistry and Morphological Sciences,University ofModena and Reggio Emilia,Modena,ItalyThe lipophilic cation JC-1(5,5ʹ,6,6ʹ-tetrachloro-1,1ʹ,3,3ʹ-tetraethyl-benzimidazolyl carbocyanine iodide)has been used for more than20yearsas a specific dye for measuring mitochondrial membrane potential( m).Inthis unit,we revise our original protocol(that made use of a single488nmlaser for the detection of monomers and aggregates,and where compensationwas an important step)to use dual-laser excitation.Moreover,thanks torecently developed multilaser instruments and novel probes for surface andintracellular markers,JC-1can be utilized by polychromaticflow cytometryto simultaneously detect,without any compensation betweenfluorescences,m along with other biological parameters,such as apoptosis and theproduction of reactive oxygen species.C 2015by John Wiley&Sons,Inc.Keywords:apoptosis r mitochondrial membrane potential r JC-1r polychro-maticflow cytometry r Annexin V r CellRoxHow to cite this article:De Biasi,S.,Gibellini,L.,and Cossarizza,A.2015.UncompensatedPolychromatic Analysis of Mitochondrial Membrane PotentialUsing JC-1and Multilaser Excitation.Curr.Protoc.Cytom.72:7.32.1-7.32.11.doi:10.1002/0471142956.cy0732s72INTRODUCTIONThe dissipation of the mitochondrial transmembrane potential( m)constitutes anearly and irreversible step in the cascade of events that,in several cell types,can lead toprogrammed cell death(apoptosis)(Galluzzi et al.,2012).Several probes are available to measure m byflow cytometry,but some of them havea low specificity for this organelle;conflicting data in the literature about the role ofm dissipation during the apoptotic process could be,at least in part,ascribed to thislack of specificity.After excitation with a blue laser at488nm,thefluorescent dye5,5ʹ,6,6ʹ-tetrachloro-1,1ʹ,3,3ʹ-tetraethyl-benzimidazolyl carbocyanine iodide(JC-1),a lipophilic cation ex-isting in a monomeric form,emits in the green region.However,in mitochondriathat have a high m,JC-1forms so called J-aggregates,described almost80yearsago(Jelley,1936),and undergoes a reversible change influorescence emission fromgreen to ing commonflow cytometers equipped with such lasers,for sev-eral years mitochondria have been studied by detecting the two emissions of JC-1by the normalfilters present in FL1(for monomers)and FL2(for aggregates)(Cos-sarizza et al.,1993;Cossarizza et al.,1995;Polla et al.,1996;Cossarizza et al.,1997;Salvioli et al.,2000;Cossarizza et al.,2002;Lugli et al.,2007;Troiano et al.,2007; Gibellini et al.,2012;Abu et al.,2014;Marringa et al.,2014;also see older version Current Protocols in Cytometry7.32.1-7.32.11,April2015Published online April2015in Wiley Online Library().doi:10.1002/0471142956.cy0732s72Copyright C 2015John Wiley&Sons,Inc.Nucleic Acid Analysis7.32.1 Supplement72of this unit at /doi/10.1002/0471142956.cy0732s41/full).Measurements using this dye provide information on changes in m(typically,adecrease in m causes a relevant shift from orange to greenfluorescence emis-sion),as well as on total mitochondrial content(based on the intensity of the greenfluorescence emission).A number of studies have since shown the superiority ofJC-1over other dyes—e.g.,rhodamine123(R123)or3,3ʹ-dihexyloxadicarbocyanineiodide[DiOC6(3)]—that were used for the same purpose,and demonstrated thatJC-1is also unaffected by changes in plasma membrane potential(Salvioli et al.,1997;Lugli et al.,2007;Troiano et al.,2007;also see older version of this unit at/doi/10.1002/0471142956.cy0732s41/full).This unit discusses a new method to detect JC-1(see Basic Protocol1),based upon theuse of two lasers,one to excite JC-1monomers(by the canonical488-nm laser line),and the other to excite JC-1aggregates(by a yellow laser emitting at561nm).Thetypical excitation by the blue laser excites JC-1with high efficiency,but sometimesrequires significant compensation between FL1and FL2.In contrast,yellow laser allowsa better resolution,and thus a clearer visualization of monomers and aggregates withoutcompensation(Perelman et al.,2012).For this reason,we have revised our basic JC-1protocol using the two different lasers quoted above.Furthermore,we have recently developed another polychromaticflow cytometric assay(see Basic Protocol2)utilizing JC-1and other probes for the simultaneous detection of m,reactive oxygen species(ROS,by CellRox DeepRed),and apoptosis(by Annexin V,detecting the exposure of phosphatidylserine on the plasma membrane).This protocolcan be applied when the simultaneous analysis of multiple parameters during apoptosisis required,e.g.,in investigating the role of certain proteins on cell phenotype or whentesting the cytotoxicity of compounds of pharmacological interest.CAUTION:For the protection of laboratory personnel from potential infectious agents (e.g.,hepatitis and HIV),handle human samples using disposable gloves in a biological safety cabinet.CAUTION:All probes described in this unit are potentially hazardous(see manufacturers’MSDSs),and users should wear gloves during the staining procedures.BASIC PROTOCOL1BASIC DETERMINATION OF MITOCHONDRIAL MEMBRANE POTENTIAL USING JC-1:DUAL-LASER EXCITATION OF THE DYEA VOIDS COMPENSATION ISSUESThis protocol is intended for cells such as peripheral blood mononuclear cells(PBMCs)or cell lines such as RKO,HL60,MCF7,and U937.Other cell types may also be stainedusing minor adjustments to the steps described below.Typically,by using a488-nm blue laser,it can be observed that cells with high m(that form JC-1aggregates)emit orangefluorescence(atß590nm);those with low m(containing JC-1in its monomeric form)emit greenfluorescence(atß520nm) (Cossarizza et al.,1993).Recently it has been demonstrated that alternative excitationwavelengths can facilitate the detection of m,and,most importantly,use of twowavelengths avoids the need for compensation.Indeed,the excitation wavelength561nm(i.e.,yellow laser)is above the emission spectra of JC-1monomers,and selectivelyexcites J-aggregates;hence there is no need to compensate green and orangefluorescence(Perelman et al.,2012).Thus,we have adapted our original protocol(that made use ofa single488-nm laser,and where compensation was an important step)to an instrumentequipped with a blue and a yellow laser(like the Attune NxT,from Life Technologies).Analysis ofMitochondrialMembranePotential UsingJC-1andMultilaserExcitation7.32.2Supplement72Current Protocols in CytometryMaterialsExperimental samples:human peripheral blood lymphocytes or monocytes,orhuman tumor cell lines(e.g.,RKO,HL60,U937,MCF7);here we use RKOcells,which derive from a colon carcinoma and grow adherent to the plasticflask Complete RPMI culture medium,1ml per sample1M valinomycin[dissolve valinomycin(mol.wt.1111.32;Sigma-Aldrich)indimethylformamide(DMF)and store in a glass container up to6months at4°C]or1mM carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone(FCCP;Sigma Aldrich)2.5mg/ml JC-1(5,5ʹ,6,6ʹ-tetrachloro-1,1ʹ,3,3ʹ-tetraethylbenzimidazolyl-carbocyanine iodide):prepare by dissolving JC-1(Life Technologies,ThermoFisher Scientific)in dimethylformamide(DMF);store in a glass container up to2years at–20°C,protected from lightPhosphate-buffered saline(PBS)3.5-ml,55×12–mm plastic tubes(Sarstedt,or equivalent)Centrifuge(Minifuge RF;Heraeus),or equivalentFlow cytometer equipped with a488-nm blue laser and with a561-nm yellow laser,e.g.,Attune NxT(Life Technologies)Additional reagents and equipment for counting(APPENDIX3A)and culturing(APPENDIX3B)mammalian cellsPrepare cells1.Count a sample of the experimental cells of interest(APPENDIX3A).This protocol can be used to stain either cells growing in suspension or adherent cellsafter they have been released from the plate by trypsinization(APPENDIX3B)and counted(APPENDIX3A).2.Collect at least2×105cells from the experimental samples in55×12–mm tubesby centrifuging5min at300×g,room temperature.Collect the same number ofcells to use for a positive control.3.Decant and discard the medium and resuspend the cell pellet in1ml fresh completeRPMI culture medium.4.For obtaining a so-called“positive control,”i.e.,a sample where all cells have de-polarized mitochondria,prepare one sample of cells treated with valinomycin(finalconcentration0.1μM)or with carbonyl cyanide p-(trifluoromethoxy)phenylhydra-zone(FCCP,final concentration250nM).Incubate10min or45min,respectively,at37°C.Drugs such as the K+ionophore valinomycin or the proton translocator FCCP are ableto collapse theΔΨm.Note that to avoid problems related to intracellular drug metabolism,in some instancesvalinomycin is preferred over FCCP or ClCCP(and is also less expensive).Stain with JC-15.Add1μl of2.5mg/ml JC-1fluorescent probe(2.5μg/mlfinal concentration)to theexperimental and positive control cells and shake the cell suspension until the dyeis well dispersed and gives a uniform red-violet color.JC-1tends to form aggregates when added to normal aqueous medium.To avoid this,add the probe while gently vortexing.6.Incubate the samples10min in the dark,37°C.Nucleic AcidAnalysis7.32.3 Current Protocols in Cytometry Supplement72Figure 7.32.1Changes in JC-1fluorescence after mitochondrial membrane depolarization in RKO cells treated with valinomycin,as described in Basic Protocol 1.Samples were acquired using 488-nm laser only (A ),or with dual-laser excitation (B ).Control cells (CTR)were stained with 2.5μg/ml JC-1.Note the shift to the bottom and to the right of cells with mitochondria depolarized by treatment with 100nM valinomycin.Right panel shows the merging of the left and center panels.Green-orange compensation was ß4%and orange-green compensation was ß10%;compensation was required to better visualize monomers and aggregates.All reagents must be at room temperature and carefully checked for pH (7.4)when used,because ΔΨm is very sensitive to alterations of these conditions.The staining procedure must be carried out away from direct intense light,and incubation must be in the dark because of the light sensitivity of JC-1.7.Wash the cells by centrifuging 5min at 300×g ,room temperature,discarding the supernatant,and resuspending the cells in 1ml PBS for analysis on cytometer.Set up flow cytometer 8.Detect JC-1fluorescence of the experimental and positive control samples using a classical green band-pass filter centered at 525/50nm for monomers detection (channel of blue laser)and a classical greenish orange band-pass filter centered at 585/42nm (usually those for a channel collecting fluorescence signals deriving from the excitation with the blue or the yellow laser).The most common flow cytometers are typically equipped with only a 488-nm argon or solid-state laser;no special requirements are needed to analyze ΔΨm .The gain of photomultipliers (PMTs)obviously depends on the cytometer used,but generally JC-1does not require any substantial increase in PMT amplification;green-orange compensation can be ß4%and orange-green compensation ß10%.However,note that no compensation is needed if a blue and a yellow laser are used to detect monomers and aggregates,respectively.See Figure 7.32.1for a typical example of JC-1staining of control (CTR)RKO cells,and of RKO cells treated with valinomycin.Detection was performed by using a single blue laser (A)or using blue and yellow lasers (B).This treatment results in a relevant change in the fluorescence distribution:cells with depolarized mitochondria can be easily identified as those going from the center of the plot to the lower right quadrant.Analysis ofMitochondrial Membrane Potential UsingJC-1andMultilaser Excitation7.32.4Supplement 72Current Protocols in Cytometry9.On the basis of the laser used,adjust the voltage of the respective PMTs to obtainthe bivariate green versus orange distributions similar to those shown in Figure7.32.1A and B,and then record the control e the same PMT settings forthe subsequent samples.Analyze JC-1stained experimental samples10.Acquire fluorescence data for experimental samples in listmode,using a log scalefor the fluorescence channels.Cells with high ΔΨm are those forming J-aggregates;thus,they show high orangefluorescence.On the other hand,cells with low ΔΨm are those in which JC-1maintains (orre-acquires)its monomeric form,and thus show green fluorescence.Once mitochondriaare depolarized,JC-1monomers redistribute in other membranous compartments withlower ΔΨ.As a consequence,the green fluorescence intensity of depolarized cells is alittle bit higher than that of polarized ones simply because of the presence of a higheramount of JC-1monomers inside the cell.11.Recommended for samples with heterogeneous cell populations:Set a gate on thepopulation of interest,then proceed with adjustment of PMTs,as well as compen-sation if a 488-nm laser is used.Dual-laser excitation of the dye does not requirecompensation.When the sample contains a heterogeneous cell population,it is possible to see differentfluorescence patterns due to different autofluorescences and the variable content in termsof membranes and mitochondria of cell subpopulations.This is the case for peripheralblood mononuclear cells (PBMCs),lymphocytes,and monocytes,the first being smallerand having fewer mitochondria than the latter.Accordingly,the fluorescence pattern ofJC-1for such a sample shows at least two distinct peaks,one corresponding to lympho-cytes,and the second,brighter in both green and orange,corresponding to monocytes.It is thus recommended to first set a gate on the population of interest,then proceed withadjustment of PMTs and compensation.BASIC PROTOCOL 2ANALYSIS OFM ,APOPTOSIS,AND REACTIVE OXYGEN SPECIESCONTENT BY 4-LASER POLYCHROMATIC FLOW CYTOMETRYThis protocol allows the analysis of m along with the detection of early apoptotic cells,and the quantification of the amount of reactive oxygen species in the cells of interest.It has been developed taking into account the possibility of simultaneously using fourlasers (by using an Attune NxT from Life Technologies)and avoiding any compensationamong dyes.Fine analysis of the apoptotic process requires the detection of multiple cell functions atthe same time,and it could be highly informative to reveal whether cells with differentm also differ with respect to other parameters.This assay is recommended whenstudying compounds that can have differential effects on the cell populations of interest.This protocol uses three different probes:JC-1(for m ),annexin V conjugated withPacific Blue (for detecting the exposure of phosphatidylserine on the plasma membrane,a well known phenomenon which identifies early apoptotic cells),and CellRox DeepRed (for measuring ROS production).CellRox is a cytoplasmic cell-permeable non-fluorescent (or very weakly fluorescent)reagent which,in a reduced state and uponoxidation,exhibits a strong fluorogenic signal.CellRox Deep Red can be excited by a638-nm laser,and emits at ß665nm.For complete information regarding the probesdescribed here,see Internet Resources at the end of this unit.Annexins are a family of soluble proteins (13different isoforms)with four to eightrepeats of a 75–amino acid consensus sequence relevant for Ca 2+binding.They areinvolved in membrane transport,regulation of protein kinase C,formation of ion channels,Nucleic Acid Analysis 7.32.5Current Protocols in Cytometry Supplement 72endocytosis,exocytosis,and membrane-cytoskeleton interactions.Annexin V binds with peculiar specificity to phosphatidylserine residues,which are precociously exposed on the external leaflet of the plasma membrane during apoptosis (Lizarbe et al.,2013).Thus,when cells are annexin V positive,they have entered into an early phase of apoptosis.The annexin V–Pacific Blue conjugate is violet excitable,making it ideal for instruments with a laser at 405nm,and for multicolor experiments that include green-or red-fluorescent dyes.The Pacific Blue-conjugated annexin V emits at ß455nm after excitation by a violet light source.Before starting with sample analysis,running samples stained with single fluorochromes (see steps below)is suggested to properly set up fluorescence levels.Note that also in this case there are no compensation requirements.Materials Cells in culture (ATCC):in suspension or adherent in 24-well tissue culture plate (as in Basic Protocol 1,we use RKO cells derived from human colon carcinoma Complete RPMI culture medium Phosphate-buffered saline (PBS)CellRox Deep Red Reagent (Life Technologies)2.5mg/ml JC-1(5,5ʹ,6,6ʹ-tetrachloro-1,1ʹ,3,3ʹ-tetraethylbenzimidazolylcarbocyanine iodide);prepare by dissolving JC-1(Life Technologies,Thermo Fisher Scientific)in dimethylformamide (DMF);store in a glass container up to 2years at –20°C,protected from light Annexin V binding buffer (see recipe)Pacific Blue-conjugated annexin V (Life Technologies,Thermo Fisher Scientific):store at 4°C,protected from light 3.5ml,55×12–mm plastic tubes (Sarstedt,or equivalent)Centrifuge (Minifuge RF;Heraeus),or equivalent.Attune NxT cytometer or equivalent cytometer equipped with four light sources for excitation at 405nm (violet laser,for Annexin V),488and 561nm (blue and yellow lasers,for JC-1),and 638nm (red laser,for CellRox)and filters for collecting fluorescence emissions at 455/40(for annexin V),520/20(for JC-1monomers),585/42(JC-1aggregates),and 660/40(CellRox)Additional reagents and equipment for counting (APPENDIX 3A )and culturing (APPENDIX 3B )mammalian cells and detaching adherent cells using trypsin (see APPENDIX 3B )Prepare cells 1.Count a sample of the cells in culture (see APPENDIX 3A ).For cells in suspension 2a.Collect at least 3×105cells from experimental samples by centrifuging 5min at 300×g ,room temperature.Collect the same number of cells to use for a positive control.3a.Decant and discard the medium and bring the total volume up to 1ml with prewarmed RPMI culture medium.For adherent cells 2b.Decant and discard the growth medium.3b.Add 1ml prewarmed culture medium (RPMI or similar)to the cells in the plate.Analysis ofMitochondrial Membrane Potential UsingJC-1andMultilaser Excitation7.32.6Supplement 72Current Protocols in CytometryThis protocol has been set up using blood cells and has been shown to work with differentcell lines.However,particular attention should be given to adherent cell lines,detachmentof which from the culture plate by trypsin-EDTA is required before cytofluorimetricanalysis.The detachment procedure could be particularly harmful to those cells thathave been damaged during the in vitro treatment,i.e.,by the presence of an apoptogenicsubstance.In this case,the multistaining procedure described here could be performedon still-adherent cells by adding the probes directly to the culture plate.Stain cells4.Add the CellROX Reagent at afinal concentration of5μM to the cells and incubatefor30min at37°C.For cells in suspension5a.To wash the staining solution from the cells,add1ml PBS,mix by shaking gently,and centrifuge5min at300×g,room temperature.Decant and discard the supernatant.Because this protocol requires many centrifugations for the cells in suspension,theauthors suggest setting the centrifugation speed as low as possible in order to avoidcellular damages due to stress.Adding10%fetal bovine serum to PBS can decrease cellloss during washing steps.6a.Resuspend the cells in1ml complete culture medium.Proceed to step7.For adherent cells5b.Decant the staining solution from the cells and wash by adding1ml PBS,swirling, and decanting.6b.Detach the adherent cells as follows.i.Trypsinize cells as described in APPENDIX3B.The minimal amount of trypsin should be used in order to avoid both cellular damage andthe presence of aggregates in the cell suspension.In fact,cell aggregates could augmentbackground or J-aggregatefluorescence.In this case,aggregates can be eliminated fromanalysis by gating on singlets,which can be identified by plotting FS-area versus FS-height.In any case,when adherent cell lines are treated with apoptogenic substances,remember that apoptotic cells spontaneously detach andfloat in the supernatant;theyshould not be discarded but collected and analyzed separately or together with attachedcells.ii.Add1ml complete culture medium to neutralize trypsin activity.iii.Centrifuge5min at300×g,room temperature,and discard the supernatant.Proceed to step7.7.Add1μl of2.5mg/ml JC-1(2.5μg/mlfinal concentration)to the pellet from step6a or6b and mix until the dye is well dispersed and gives a uniform red-violet color.Incubate the samples10min in the dark,room temperature.JC-1tends to form aggregates when added to normal aqueous medium.To avoid this,add the probe while gently vortexing.8.Wash with1ml PBS as in step5a or5b.9.Resuspend the cells in195μl annexin V binding buffer.10.Add5μl of Pacific Blue–conjugated annexin V(at concentration provided by themanufacturer)and incubate15min at room temperature.Staining with annexin V is the last step of the protocol because annexin V binding tophosphatidylserine is affected by the presence of its incubation buffer.In the authors’Nucleic AcidAnalysis7.32.7 Current Protocols in Cytometry Supplement72experience,washing or resuspending cells with PBS causes annexin V detachment from phosphatidylserine.11.Resuspend the cells in1ml annexin V binding buffer.Acquire samples on cytometer12.First acquire blank samples and cells without CellRox,to set the level of backgroundfluorescence for the Alexa647channel.This type of analysis requires aflow cytometer equipped with three light sources and appropriate collectionfilters for all the dyes(see Materials list).13.Acquire at least30,000total events.Analyze data14.Identify cell populations on the basis of annexin V,i.e.,live(Annexin V–),apoptotic(Annexin V+).Analyze m and ROS content in these subsets.Since multiple parameters are simultaneously analyzed,different techniques for data interpretation can be adopted depending on the user’s interests.In this case,should the researcher be interested in detectingΔΨm and ROS production in early apoptotic or healthy cells,a gate can be designed on annexin V positive or negative cells,where the other parameters are thus analyzed(see Fig.7.32.2).REAGENTS AND SOLUTIONSUse deionized,distilled water in all recipes and protocol steps.Annexin V binding buffer0.477g HEPES(10mM)1.636g NaCl(140mM)0.073g CaCl2(2.5mM)H2O to200mlAdjust pH to7.4and store up to1year at4°CMilli-Q-purified(double purified)water may also be used in this recipe. COMMENTARYBackground InformationMitochondria play an active role in theregulation of programmed cell death,and in-deed the collapse in m can occur duringthe apoptotic process(Green et al.,2011).The opening of the mitochondrial permeabilitytransition pore—a mitochondrial protein com-plex formed by the adenine nucleotide translo-cator(ANT),the voltage-dependent anionchannel(VDAC),and the peripheral benzo-diazepine receptor(PBR)—can induce loss of m,release of apoptogenic factors,and loss of oxidative phosphorylation(Martel et al.,2014).However,whether loss of m is acause or a consequence of the triggering ofapoptosis still remains a matter of debate.De-pending on the apoptotic model used,loss of m may be a late(Cossarizza et al.,1994)or an early(Zamzami et al.,1995)event.More-over,loss of m is responsible for the release of apoptosis-inducing factor(AIF),which con-sequently translocates to the nucleus and pro-motes chromatin condensation and fragmen-tation(Kroemer et al.,2007).Other mecha-nisms initiating apoptosis(e.g.,cytochromec release or activation of executioner cas-pases)are independent of the disruption of m(Kluck et al.,1997;Bossy-Wetzel et al., 1998).Several techniques are used to investigatethe role of this organelle,including classicalbiochemical or molecular biology methods;flow cytometry clearly represents the mostrapid and powerful tool for investigating m at the single-cell level.Many probes are available for this purpose,but some of them, e.g.,R123and DiOC6(3),are not fully adequate(Salvioli et al.,1997).As a consequence,discrepancies in the data regarding the role of m in the regulation of the apoptotic process may be also attributed to the use of inappropriate probes.A detailed analysis of other dyes is reported in UNIT9.14 (Cossarizza and Salvioli,2000).Analysis ofMitochondrialMembranePotential UsingJC-1andMultilaserExcitation7.32.8Supplement72Current Protocols in CytometryFigure7.32.2Multilaser,uncompensated analysis of apoptosis,mitochondrial membrane potential,and production of reactive oxygen species.RKO cells were cultured in the absence(A)or presence(B)of H2O2(1hr)and(C)5μM CDDO (24hr).Cell were stained as described in Basic Protocol2.Viable and apoptotic cells were identified by positivity for annexin V; m was analyzed by JC-1,ROS production by CellRox Deep Red.We have demonstrated that JC-1is an excel-lent potentiometric probe,having the peculiar ability to change color reversibly depending on the m.This property is due to the reversible formation of JC-1aggregates upon polariza-tion of mitochondrial membrane,which causes a shift in emitted light fromß530nm(emis-sion of monomers)toß590nm(emission of J-aggregates).In living cells,the color of the dye changes reversibly from green to orange as the mitochondrial membrane becomes more polarized(Reers et al.,1991).Aggregate for-mation begins at potential values on the order of80to100mV,and reaches the zenith at ß200mV.When488nm was the sole available laser line,researchers had to cope with compen-sation,which had to be set up considering the spillover of the twofluorescences,and re-quired not only the preparation of“biologi-cal negative controls”(i.e.,samples of cells treated with a depolarizing agent to see the area where cells with a low m tended to go),but also a certain experience on the part of the operator.In any case,excitation with 488-nm laser was quite efficient and allowed,and is currently allowing,a significant num-ber of studies.Modernflow cytometers havemore excitation sources than in the past.Themain advantage of a second excitation sourcefor JC-1aggregates is well evidenced by factthat compensation is no longer needed,sinceyellow laser does not excite JC-1monomers(Perelman et al.,2012).JC-1staining can be combined with multi-ple probes in a polychromaticflow cytometricassay to detect changes in m together withother parameters during apoptosis;Basic Pro-tocol2can be useful and informative,becauseseveral cell functional subsets with different characteristics can be simultaneously identi-fied in a given population.This makes it pos-sible not only to discriminate cell death,butalso to investigate whether similar compoundsexert differential effects in the same cell type.This type of analysis,combined with high-throughput technologies,could be adopted forthe screening of the toxicity of a variety of compounds,in order to obtain multiple infor-mation about the investigated molecules.Nucleic AcidAnalysis7.32.9Current Protocols in Cytometry Supplement72。

第48卷第7期2020年7月硅酸盐学报Vol. 48，No. 7July，2020 JOURNAL OF THE CHINESE CERAMIC SOCIETY DOI：10.14062/j.issn.0454-5648.20200066 基于复合固体聚合物电解质的固态钠电池张强强1,2，苏醒1,2，陆雅翔1,2，胡勇胜1,2(1. 中国科学院物理研究所，北京 100190；2. 中国科学院大学，北京 100049)摘要：将钠离子导体Na-β’’-Al2O3作为活性填料引入聚氧化乙烯(PEO)/双(三氟甲基磺酰)亚胺钠(NaTFSI)固体聚合物电解质(SPE)中，得到有机–无机复合固体聚合物电解质(CPE)。

对SPE及CPE的相结构、相转变、离子电导率、离子迁移数及电化学稳定性等基础理化性质进行了表征分析，对两者在固态钠电池Na3V2(PO4)3@C||Na中的电化学性能进行了测试。

结果表明：Na-β’’-Al2O3的引入，有效提升了钠离子迁移数(SPE为0.19 ，CPE为0.71)和钠离子电导率(80℃时，SPE为1.65×10–4 S/cm，CPE为8.19×10–4 S/cm)。

基于CPE的固态钠电池表现出更加优异的循环稳定性，0.5C循环100周后容量保持率为93.9%，2.0C循环500周后容量保持率为74.0%。

关键词：聚氧化乙烯；双(三氟甲基磺酰)亚胺钠；固体聚合物电解质；复合固体聚合物电解质；钠电池中图分类号：TQ175 文献标志码：A 文章编号：0454–5648(2020)07–0939–08网络出版时间：2020–04–14A Composite Solid-state Polymer Electrolyte for Solid-state Sodium BatteriesZHANG Qiangqiang1,2, SU Xing1,2, LU Yaxiang1,2, HU Yongsheng1,2(1. Institute of Physics, Chinese Academy of Sciences, Beijing 100190, China;2. University of Chinese Academy of Sciences, Beijing 100049, China)Abstract: An organic-inorganic composite polymer electrolyte (CPE) was constructed via introducing an active filler of Na-β’’-Al2O3 into a solid-state polymer electrolyte (SPE) composed of poly(ethylene oxide) (PEO) and sodium bis(trifluoromethanesulfonyl)-imide (NaTFSI). The phase composition, ionic conductivity, Na+ transference number, and electrochemical stability were investigated. The electrochemical properties of SPE and CPE in Na||Na3V2(PO4)3@C solid-state sodium batteries were obtained. The results show that the CPE has a greater Na+ transference number of 0.71 than SPE (i.e., 0.19) and a higher Na+ conductivity of 8.19×10–4 S/cm than SPE (i.e., 1.65×10–4 S/cm) at 80℃. The CPE exhibits a superior cycling stability with capacity retentions of 93.9% after 100 cycles at 0.5C and 74.0% after 500 cycles at 2.0C in Na|CPE|Na3V2(PO4)3@C solid-state batteries.Keywords: poly(ethylene oxide); sodium bis(trifluoromethanesulfonyl)imide; solid-state polymer electrolyte; composite polymer electrolyte; sodium batteries随着社会的发展和对能源的巨大需求，人类对能源材料的开发不断增长。

陈林林，范天娇，李伟，等. 金纳米粒子修饰电极循环伏安法测定食品中的碘[J]. 食品工业科技，2022，43（1）：288−294. doi:10.13386/j.issn1002-0306.2021030386CHEN Linlin, FAN Tianjiao, LI Wei, et al. Determination of Iodine in Food by Gold Nanoparticles Modified Electrode Cyclic Voltammetry[J]. Science and Technology of Food Industry, 2022, 43(1): 288−294. (in Chinese with English abstract). doi:10.13386/j.issn1002-0306.2021030386金纳米粒子修饰电极循环伏安法测定食品中的碘陈林林1,*，范天娇1，李　伟1，郑凤鸣1，杨茜瑶1，张佳欣1，辛嘉英1,2（1.哈尔滨商业大学食品工程学院, 黑龙江哈尔滨 150028；2.中国科学院兰州化学物理研究所羰基合成与选择氧化国家重点实验室, 甘肃兰州 730000）摘　要：为寻求一种快速、简便、灵敏的食品中碘的测定方法，利用循环伏安法（CV ）构建金纳米粒子修饰电极检测碘离子（I -）体系。

利用甲烷氧化菌素（Mb ）原位还原纳米金（Mb@AuNPs ），电沉积法制备自组装修饰电极。

通过透射电子显微镜对Mb@AuNPs 表征，CV 考察碘离子的电化学行为。

确定碘离子检测的优化条件为：电沉积扫描速率0.11 V/s 、扫描圈数30圈、缓冲溶液浓度0.05 mol/L 、缓冲溶液pH6.5。

氧化峰电流与I -浓度在0.01~10.00 μmol/L 范围内有良好的线性关系，R 2为0.9992，检出限为2.88 nmol/L （S/N ），定量限为9.60 nmol/L ，该方法检测不同食品中碘含量的加标回收率为96.22%~103.57%。